Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. Last year we undertook to identify the unknown photopigment that mediates photoentrainment, the process by which light resets the endogenous clock, in collaboration with Mark Rollag (USUHS) and Maribeth Eiden (NIMH). We plan to construct and use retroviral vectors carrying sense and antisense versions of candidate photopigments to test the effects of over- and under-expression of these proteins on the responses of chick pineal cells to light. Melanopsin and pinopsin are each novel photopigments present in chick pineal cells and the best candidates for the photopigments mediating photoentrainment. Last year, experiments transducing GFP into the cells were successful as proof of principle and we constructed the plasmid containing the melanopsin gene. This year we cloned the previously identified pinopsin gene and inserted it into the plasmid with which we will construct the transducing vector. With the help of a commercial contractor we have synthesized peptides from melanopsin and pinopsin and are generating antibodies to assess the efficiency of transduction and the expression (or reduction in expression) of the proteins. We are about to construct the vectors, test and optimise transduction efficiency and expression (using GFP-containing and "empty" vectors as controls), and proceed with the physiologic assessment of transduction effects. We are also attempting to identify the unknown signal transduction pathway from the photopigment to the clock. We have undertaken to determine whether MAPK pathways play a role in photoentrainment by examining the effects of agents known to affect MAPK activity on circadian phase. Last year we found that PD 98059, a specific inhibitor of MAPK activation, does induce light-like phase shifts in the melatonin rhythm. This year, however, we found that U0126, another specific inhibitor, does not. Both drugs reduced phospho-MAPK levels, the active form of the enzyme assessed by immunoblots, by 70-100%. Changes in phospho-MAPK in the other direction (increases) were evoked by addition of cycloheximide and chicken serum, respectively. Again, one agent (cycloheximide) induced phase shifts while the other did not. We also examined agents (including light) that are known to induce phase shifts in the melatonin rhythm for effects on phospho-MAPK levels. Both high salt and caffeine induce light-like phase shifts. One of these (caffeine) lowered phospho-MAPK levels while the other had no effect. Taken together, these results indicate no predictable relationship between changes in phospho-MAPK and the induction of phase shifts. Finally, we found no effect of light on, and no diurnal changes in, phospho-MAPK levels. We are checking the distribution and responsiveness of MAPK among the cell types present in our cultures before concluding that changes in MAPK activity are neither necessary nor sufficient to induce phase shifts.